Epidermal Growth Factor (EGF) is a single chain polypeptide having molecular weight of approximately 62 kDa and comprised of 53 amino acid residues including three disulphide bonds. Epidermal Growth Factor (EGF) is one of the most potent, biologically available entities that play a very important role in wound healing. It binds specifically to the EGF receptors present on the cell surface, thus triggering complex biochemical mechanisms in the cells to trigger the growth. EGF is biologically unstable and physiologically nonhomogenous, particularly in the presence of moisture. EGF has a short life span of about an hour whereas DNA synthesis at the site of the wound requires about eight to twelve hours. EGF exhibits loss of biological activity due to denaturation, decomposition, condensation and precipitation due to proteolytic enzymes. This is disadvantageous because such loss of activity makes it impractical to store aqueous preparations of epidermal growth factor for extended periods of time. The use of EGF formulations for wound healing is known in the art. To over come this problem and to provide desired effective wound healing, it is reported that Egf has to be continuously applied in initial stages of healing. As a result, many formulations have been developed to increase the stability of EGF.
The prior art known to the inventors include CN 1515315 that relates to a stable synergistic composition containing EGF and bletilla extract.
U.S. Pat. No. 4,944,948 suggests a liposome gel formulation for the delivery of hEGF to the wound site to overcome the above-mentioned problems.
EP 0312208 discloses using various water-soluble or water-swellable carriers for slow release formulations for EGF. This enables release of EGF for 12 hours or more. However, these formulations are unsuitable for industrial application due to poor shelf life.
U.S. Pat. No. 4,717,717 advocates using cellulose derivative together with EGF to enhance stabilization.
EP 0312208 ('208) discloses aqueous gel formulation of EGF for controlled delivery of the active ingredient employing various water soluble polymer as a base for providing viscosity ranging from 1000 to 12,000,000 cps at room temperature.
U.S. Pat. No. 4,944,948 describes gel formulation of EGF using neutral phospholipids, negatively-charged phospholipids, and cholesterol.
Though these formulations are capable of continuously releasing EGF for 12 hours or more they are unsuitable for industrial manufacture being unstable in long term storage. Further, due to high viscosity of the formulation disclosed in '208 it forms barrier for migration of epidermal cells. Additionally, it poses problems in application at delicate wound sites.
U.S. Pat. No. 5,130,298 and EP publication No. 398615 teaches mixing EGF with metal cation for preventing degradation of EGF through ionic binding. This increases the stability of EGF to about 2 months at 4° C. This formulation also proves to be unsuitable for industrial application.
U.S. 2003050238 advises using acidic polymer such as carboxyvinyl polymer as a base to solve the above-mentioned problems of shelf life and unsuitability for utilizing in industrial fields. The patent claims to have increased the shelf life.
PCT Application No. WO99/44631 attempts to extend shelf life of hEGF. However, none of these solutions were found successful and the stability of the enzyme remains a problem as far as it stays in aqueous environment above 0° C.
It is also noticed that EGF degrades over time to form multiple species of the EGF molecule, which are believed to be degradation products. Such degradation occurs naturally as a result of environmental factors such as light, which can cause photo-oxidation; changes in pH; changes in ionic strength; changes in temperature; and physical manipulation of the molecule. This reduces the shelf life of an EGF formulation over extended storage.
Hence, there is an imperative need to develop a stable formulation of EGF. The inventors after conducting considerable research have developed EGF composition having stability over two years while maintaining its efficacy. Further, it provides public with useful choice. After prolonged research it has been found out that the compositions of EGF particularly rEGF, when prepared using physiologically acceptable agents comprising charged protein stabilizers specifically in combination with preservative, thickening agent, pH regulating agent and a carrier proves to be stable for about 2 years.